Method for linking molecular substances

ABSTRACT

The invention relates to a method for linking two or more molecular substances, by means of adapter segments, which bring about a targeted interaction based upon the affinity of proline-rich amino acid sequences and protein domains of the type WW.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/129,315 filed Nov. 4, 2002, which is a US National Phase ApplicationUnder 35 U.S.C. 371 of International Application No. PCT/EP2000/010873filed Nov. 3, 2000, which claims the priority of German Application No.19952956.6 filed Nov. 3, 1999, and the contents of each of which areincorporated herein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

The present invention concerns a process for the connection of two ormore molecular substances by adapter segments which cause a directedinteraction, based on the affinity of proline-rich amino acid sequencesand protein domains of the type WW.

The interaction of two or more molecular substances is a frequentproblem within the realm of biotechnological and pharmaceutical-medicalresearch, development, and application. In particular, the interactionsof two or more proteins or peptides as molecular substances are usuallyconsidered thereby. Such interactions are often explored as part ofbiochemical and cellular biological research, for instance, as withintra- and intercellular communication, signal transduction on amolecular level, or analyses of protein-protein interactions (amongstothers, in the usage of two-hybrid systems and processes derivedtherefrom). Moreover, the association of biomolecules, particularly oftwo or more proteins, for in vitro synthesis of fusion proteins is ofgreat importance for many biotechnological processes. Fusion proteinsgenerated in such a way can be, for instance, heterobifunctional(bivalent) antibodies (so-called diabodies; see O. Perisic, P. A. Webb,P. Holliger, G. Winters & R. L. Williams, Crystal structure of adiabody, a bivalent antibody fragment, Structure 2, pp. 1217-1226,1994), which comprise the binding domains (Fab/Fv/scFv fragments) of twodistinct antibodies. If thereby both valences are, for instance,directed respectively at tumor cells or natural killer cells, then thebivalent, hybrid fusion protein can accordingly mediate an attachment ofkiller cells onto tumor cells. In the case of immunotoxins, antibodiesare coupled with toxic substances and the cytotoxin is directed throughspecific antigen-antibody interaction into predefined cell types (see M.A. Ghetie & E. S. Vitetta, Recent developments in immunotoxin therapy,Curr. Opin. Immunol. 6, pp. 707-714, 1994).

With the help of fusion constructs and assemblates of various proteins,fundamentally any effectors can be combined with each other, and throughappropriate interactions with antigens or other biological effects, twofunctions or characteristics can be achieved in a hybrid molecule. Aseries of examples hereof are published (J. P. McGrath, X. Cao, A.Schutz, P. Lynch, T. Ebendal, M. J. Coloma, S. L. Morrison & S. D.Putney, Bifunctional fusion between nerve growth factor and atransferrin receptor antibody, J. Neurosci. Res. 47, pp. 123-133, 1997;J. M. Betton, J. P. Jacob, M. Hofnung, J. K. Broome-Smith, Creating abifunctional protein by insertion of beta-lactamase into themaltodextrin-binding protein, Nat. Biotechnol. 15, pp. 1276-1279, 1997;Y. Maeda, H. Ueda, T. Hara, J. Kazami, G. Kawano, E. Suzuki & T.Nagamune, expression of a bifunctional chimeric protein A-Vargulahilgendorfii luciferase in mammalian cells, Biotechniques 20, pp.116-121, 1996; W. Wels, I. M. Harwerth, M. Zwickl, N. Hardman, B. Groner& N. E. Hynes, Construction, bacterial expression and characterizationof a bifunctional single-chain antibody-phosphatase fusion proteintargeted to the human erbB-2 receptor, Biotechnology (N.Y.) 10, pp.1128-1132, 1992).

Heterobifunctional constructs are frequently produced through synthesisof fusion proteins at the gene level. This generally presupposessuitable connection elements (linkers) between both partners, as well asaccessible termini of the polypeptide chains. In unfavorable cases,fusion of the partners can lead to the product of the fusion beinginactive, for example because the fusion protein cannot develop acorrect three-dimensional folding topology. Thus, it is often desirablethat the connection of both fusion partners occurs in vitro, that is,after the separate synthesis and folding of both partners. Such aprocess would also allow, for instance, the fast production and analysisof various combinations of single components, without requiring newgenetic constructions each time. For the fusion of these components,adapter segments are necessary, through which the process of fusion ordirected association of the partners involved is isolated from theirproduction. Furthermore, it is thereby necessary that the adaptersegments (domains or peptide sequences) are locked onto the involvedpartners firmly without otherwise changing their specificcharacteristics.

In other applications, it is desired that a short-term, but strong,interaction results between two molecular species. Thereby, peptides andsmall protein domains play an especially important role, since in theprocess of recombinant production of proteins, they can be placedcomparatively easily on the desired target proteins. Applications ofthis are, for instance, the purification of recombinantly producedproteins through specific binding segments. Often, a polyhistidinepeptide segment is utilized for binding to nickel chelate columns (seeP. Hengen, Purification of His-Tag fusion proteins from Escherichiacoli, trends Biochem. Sci. 20, pp. 285-286, 1995), or the binding of apeptide segment known as Strep-Tag to Streptavidin (T. G. Schmidt, J.Koepke, R. Frank& A. Skerra, Molecular interaction between the Strep-tagaffinity peptide and its cognate target, streptavidin, J. Mol. Biol.255, pp. 753-766, 1996). The His-Tag process has the disadvantage,however, that the polyhistidine peptide segment can only bind tostructures containing nickel ions; the connection, for instance, of twonatural proteins or peptides is not possible in this way. For theconnection of molecular substances, the process is hence not, or only inexceptions, suitable. In preparations that are purified in this manner,one also often finds nickel ions in the solution, which makes the systemunattractive for medical-therapeutic applications. With the Strep-Tagprocess, the region of the binding partners that mediates binding isrelatively large, so that for steric reasons it is not suitable for manyconnections. Additionally, avidin and streptavidin each possess fourbinding sites, so that a regulated formation of two different linkedmolecular substances in solution is very difficult.

Apart from use in the purification of proteins labeled in such a way,the immobilization of proteins on a solid, inert matrix is also of highbiotechnological significance, for instance with the refolding ofproteins on a matrix for the prevention of aggregation processes duringfolding (see G. Stempfer, B. Höll-Neugebauer & R. Rudolph, Improvedrefolding of an immobilized fusion protein, Nat. Biotechnol. 14, pp.329-334, 1996), or the immobilization of an enzyme in a bioreactor.Polyionic sequences which have been used up to this time in theaforementioned process have the disadvantage, however, that theirinteraction is significantly disturbed by the presence of polyions, forinstance DNA in the solution, or also through various solvent additives.

The purpose of the invention at hand is to provide a process for theconnection of molecular substances that does not exhibit the mentioneddisadvantages of the current technology.

This is accomplished in accordance with the invention through a processbased upon claim 1 for the connection of two or more molecularsubstances with each other across adapter segments, distinguished inthat

one of the molecular substances is modified in such a way that as anadapter segment it displays, in at least one area, a WW domain or astructure derived therefrom,

another molecular substance is modified in such a way that as an adaptersegment it displays, in at least one area, a proline-rich sequence whichbinds at the WW domain or a structure derived therefrom,

and the molecular substances, through the association of WW domains orstructures derived therefrom and of a proline-rich sequence, come intointeraction with each other in order to achieve binding of one another.

Advantageous forms of execution emerge in the secondary claims anddescription.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are referred to in the description and theexamples.

FIG. 1 shows a schematic representation of the invention. Adaptersegments based on the interaction of proline-rich substances with WWdomains and forms derived therefrom are employed. (a) linkage of twomolecular species A and B through adapter segments. (b) linkage of twomolecular species A and B, analogous to (a), however with additionaldisulfide bridging for covalent linkage of the partners. (c)matrix-immobilization of a molecular substance through the adaptersegments (one the molecules represents the matrix or a part of thematrix). The adapter segments can be attached to the molecules at theends (termini) as well as in the form of insertions.

FIG. 2 shows in (a) a comparison, by means of SDS-PAGE, of the proteinmasses and the purification efficiencies of different variants ofpolyoma virus protein VP1, the PyVP1-CallS-T249C variant (comparable tothe wild type of the protein) and the PyVP1-WW150 variant, in which a WWdomain is inserted in a loop in the vicinity of amino acid position 150.Production and purification of the variants is comprehensively describedin example 1. With both variants, degradation products of the proteinthat exhibit a smaller molecular mass are usually noted to appear. (b)circular dichroism spectra (CD) of the PyVP1-WW150 variant and thePyVP1-CallS-T249C variant. The inserted WW domain at position 150exhibits a native folding, whereby the β-sheet proportion in theCD-spectrum rises.

FIG. 3 shows the binding of PyVP1-WW150 on a sensor chip with animmobilized proline-rich peptide, according to example 2. The threemeasurements, based on surface plasmon-resonance, show that the solventadditives exert only minor influence on affinity and specificity of theinteraction, with the additives used in (b) and (c) each representingcomplex physiological mixed substances. (a) binding of PyVP1-WW150 tothe sensor's surface under normal solvent conditions. (b) binding ofPyVP1-WW150 to the sensor's surface under application of Dulbecco's PBSas running buffer. (c) binding of PyVP1-WW150 to the sensor's surfacewith addition of fetal calf serum (FCS) as a model for the mixture ofbiologically relevant substances.

FIG. 4 shows an SDS gel for illustration of the specific binding ofPyVP1-WW150 to a matrix containing proline peptide. Lane 1: applicationVP1-WW150 (purified as in example 1); lane 2 and lane 3: different washfractions; lane 4 and lane 5: elution fractions with 1% SDS in theelution buffer; lane 6: 10 kDa molecular mass marker. The example showsthat WW domain-containing proteins can be reversibly immobilized on amatrix. The detected double band of the PyVP1 variant depicts the nativeprotein, as well as a proteolytic degradation product of the protein,which usually appears in all preparations.

FIG. 5 shows a gel filtration (TSK gel G5000PWXL, TosoHaas) for thedemonstration of the binding of a proline-rich peptide to the surface ofa virus-like capsid, with the WW domains inserted into VP1 and exposedon the surface of the capsid. Assembly of the PyVP1-WW150 protein into acapsid takes place under the conditions indicated in example 4. Aproline-rich peptide can be bound onto the virus-like capsids, which isverified through the specific absorption of a dye coupled to it. Leftabove: Proof of capsid formation through absorption at 260 and 280 nm;the capsids elute at a volume of 6 to 8 ml, the non-assembled, freepentamers appear at 9 to 10 ml. Left below: Absorption of thefluorescence-labeled peptide at 490 nm; the elution takes place parallelto the capsid elution and the pentamer elution at 6 to 10 ml. Above 10ml, surplus free peptide elutes along with the fluorescence dye. Rightabove: The free, unbound peptide shows no interaction with the matrixand elutes exclusively above 10 ml. Right below: Superimposition of thechromatograms from the left above and the left below, for illustrationof the coelution of the bound peptide with the capsid fraction.

FIG. 6 shows in (a) the purification of variants PyVP1-3C-WW1 andPyVP1-3C-WW[N-14]. The SDS gel (12%) exhibits the PyVP1 protein withoutWW domain (lane 2), the PyVP1-WW150 variant from example 1 (lane 3), aswell as both variants from example 8 (PyVP1-3C-WW1 on lane 4 and lane 5,PyVp1-3C-WW[N-14] on lane 6). Lane 1 and lane 7, standard molecular mass(10 kDa ladder). (b) purification of the GFP variant GFP-PLP, andillustration on an SDS gel (15%). Lane M, molecular weight marker (10kDa ladder); lane Int, wash fraction of the intein affinity column;fractions 1 to 9, various elution fractions the GFP-PLP-Proteins.

FIG. 7 shows the packaging of molecular substances in the interior ofvirus-like coats on the basis of polyoma virus VP1 variants. (a)wrapping of GFP-PLP in coats which contain PyVP1-3C-WW1. GFP-PLP isadded before assembly under standard conditions in six-fold molarexcess. A gel filtration experiment (TSK gel G6000PWXL, TosoHaas) isshown in which capsid fractions (elution at 9 ml) are detached fromfree, non-assembled pentamers of PyVP1 variants as well as from the GFPprotein (11 to 13 ml). A detectable quantity of GFP, which was directedinto the interior of the capsid through the WW domain/polyprolineinteraction, is present in the capsid fraction. (b) encapsidation of GFPinside of virus-like capsids with WW domain at the N-terminus, assembledfrom PyVP1-3C-[N-14]-PLP (proline-rich sequence at the shortenedN-terminus). Analogous to the example in (a), GFP-WW1 is incubated withPyVP1-3C-[N-14]-PLP and capsids are produced through assembly understandard conditions. The polyproline peptides, through affinity to theWW domain, are thereby brought into the interior of the capsids. (c)encapsidation of a fluorescence-marked peptide (proline-rich sequence)in the interior of virus-like capsids. Analogous to the example in (a),the peptide is incubated with PyVP1-3C-WW[N-14] and capsids are producedthrough assembly under standard conditions. The polyproline peptides,through affinity to the WW domain, are thereby brought into the interiorof the capsids. (d) wrapping of GFP with proline-rich sequence at theC-terminus inside of virus-like capsids, which are assembled withPyVP1-3C-WW[N-14]. Analogous to the example in (a), GFP-PLP is incubatedwith PyVP1-3C-WW[N-14] and capsids are produced through assembly understandard conditions. The GFP-PLP, through affinity to the WW domain, isthereby brought into the interior of the capsids.

FIG. 8 shows an SDS gel for illustration of the purification of proteinswith WW domain from a mixed substance, here a cellular extract. Lane 1:10 kDa molecular weight marker; lane 2: crude extract of(PyVP1-3C-WW1)-intein-CBD fusion protein; lane 3: run-through; bands 4to 10: various fractions of the elution of the fusion protein, with 2%SDS in the elution buffer. Immobilization of the fusion protein heretakes place through a column with a covalently bound proline-richpeptide. After application of the crude extract, the column is washedwith a total of 10 column volumes of a buffer that contains 2 M NaCl.Besides the fusion protein, degradation products therefrom are detected,as well as molecular chaperones, which are known to bind to PyVP1.

FIG. 9 shows the disulfide bridging of a molecular substance with a WWdomain, which was fused to Glutathion-S-Transferase (GST) for thepurpose of affinity purification. For bridging, two variants of a WWdomain were used, at which an amino acid was exchanged for a cysteine atone position of each. That is, for one, the variant D8C, on which theaspartate was exchanged at position 8 in the WW domain for cysteine and,for another, K19C, at which Lysine19 was replaced with cysteine. Themolecular substance here is a proline-rich peptide with the sequenceCSGP₈LP (SEQ ID NO:15), which was marked with a fluorescence dye (OregonGreen, OG, Firma Molecular Probes) for the purpose of the analysis atthe amino group of the N-terminus. The disulfide bridging was performedas described in example 7. In order to analyze the bridging, the samplewas subjected to a reversed-phase HPLC (HPLC column: YMC protein-Rp C₁₈:running buffers A: 0,1% TFA in H₂O, running buffer B: 80% ACN, 0.1%TFA). According to these chromatographs, WW domain and free, non-bridgedpeptide could be separated from one another (elution times: peptide 12min., WW domain 25-27 min.), while disulfide-bridged peptide and the WWdomain almost coelute (28 min). By means of the fluorescence label ofthe peptides, the peptide can be detected along with the WW domain. FIG.9( a) shows that this was the case for the WW domain-variant K19C, nothowever for D8C. The cause for this can lie in the stericinaccessibility of the cysteines of variant D8C. As proof of theWW-domain specificity of this bridging, the cysteine-free variant of theWW domain was analyzed in a parallel experiment, which likewise showedno bridging. FIG. 9( b) shows that the covalent interaction of the WWdomain variant K19C and proline-rich peptide can be broken by additionof a reduction agent (50 mM DTT). The fluorescing peptide exists againthereafter completely in free form.

DETAILED DESCRIPTION OF THE INVENTION

The linkage of two or more different molecular substances (molecularspecies) into one—usually heterobifunctional—fusion construction is aprocess of high biotechnological and pharmaceutical interest. Usually,as part of the invention-compliant application, proteins and/or peptidesare used as the molecule species to be joined, since the adaptersegments of the invention at hand originate from this chemical class.According to the invention, other molecular substances that possess oneof the adapter segments of this invention are also usable. For instance,in compliance with the invention, a solid matrix can be loaded with amolecular substance over the specified adapter segments. Often, bothsubstances to be joined must be stably and covalently linked with eachother. Conversely, for some applications it can also be desired that theinteraction between both molecular species exists only for a limitedtime and can be quickly dissolved again, for instance through extraneousadditives. In yet other applications, a molecule species must beimmobilized for a limited time, hence interact in a specific way with amatrix, for instance for the purification of a protein from a crudecellular extract in the recombinant production of a protein, or for amatrix-supported refolding of the protein. For such applications, theinvention at hand is appropriate.

Compliant with the invention, a protein can, for instance, be directedinto the interior of a virus-like coat for wrapping, or two or moredifferent proteins can be joined into a chimeric protein with newcharacteristics, for instance as bivalent antibodies. Analogously, thisinteraction can be used for the immobilization of a molecular species,for instance for the separation of this substance from a mixture ofsubstances.

In addition to connection through adapter segments, which is based onthe interaction between WW domain and proline-rich sequence, covalentlinkage of the molecular substances with each other can take place. Thiscovalent linkage, through disulfide bridging via cysteines artificiallyintroduced at a suitable spatial site in both molecular substances, canthereby lead to a durable connection between both molecular substances.Via disulfide bridging, bifunctional fusion molecules can be generatedwhich exist stably under physiological and all ordinary solventconditions, and are thus also useful for medical, therapeutic,diagnostic, and biotechnological processes.

Possible application forms of the invention as described above are alsopresented in exemplary manner in FIG. 1.

For the connection of two or more molecular substances in compliancewith the invention, the highly specific interaction of protein segmentsknown under the term WW domain with a proline-rich peptide sequence(with a proline content of more than 50% within a short peptidesuccession of 2 to 6 amino acids) is exploited. These two molecularspecies show an unusually strong interaction with each other (K_(D) 20to 100 nM), when they are incubated together. The slow dissociation ofthe partners leads to the fact that the interaction is at first onlytemporarily effective. If this is unwanted, the dissociation can beprevented through the fixation of the binding partners by means of adisulfide bridge. Cysteines are artificially introduced in suitablespatial position into both adapter segments or within the region of theadapter segments. After association of the partners, the cysteine pairscan be oxidized through suitable choice of redox conditions and are, inthis way, durably combined covalently with each other. The emerginghybrid fusion protein can display essential characteristics of therespectively underlying molecule species.

The WW domain is a small, globular protein domain, which usuallyconsists of 30 to 40 amino acids (see M. Sudol, The WW Domain BindsPolyprolines and is Involved in Human Diseases, Exp. & Mol. Medicine 28,pp. 65-69, 1996), yet shorter variations are also known. WW domainsdisplay a high natural affinity to proline-rich ligands, which are boundwith dissociation constants of 20 to 100 nM. The proline-rich ligandspossess the minimum length necessary for binding of 5 to 15 amino acidswith a proline content of more than 50% within this segment, whereby thedirect interaction usually appears within a local segment of 2 to 6amino acids (with more than 50% proline content). Natural ligands arethereby almost exclusively proteins that contain proline-rich segmentsin their amino acid sequence, however proline-rich peptides are alsospecific ligands of WW domains.

The designation WW domain derives from the observation that twoconserved tryptophan residues (abbreviated WW) appear with a spacing of20 to 22 amino acids; the second tryptophan, and a series of chieflylikewise-conserved hydrophobic amino acids, thereby form the bindingpocket for the proline-rich ligands. A conserved proline is oftenlocated with a spacing of 2 amino acids after the second tryptophan. Aseries of different WW domain-types are known, which are presentlyarranged in 4 classes and which distinguish themselves from one another,particularly in reference to the preferentially-bound peptic ligands. WWdomains can, in principle, compete with the (structurally unrelated)SH3-domains for the binding of proline-rich ligands, yet the ligands ofthe SH3-domains display deviant consensus sequences, so thatproline-rich peptide ligands can be derived, which are specificallybound by WW domains. Furthermore, the binding of WW domains toproline-rich ligands is usually stronger than that of SH3 domains. Thefollowing table gives an overview of types and ligand-binding qualitiesof the of WW domain proteins.

WW Specific binding motif domain type of the proline-rich ligands*Example/Agent Type I Pro-Pro-(arbitrary)-(Tyr) YAP65, Pin1, DystrophinType II Pro-Pro-Leu-Pro (SEQ ID FBP11, FE65 NO: 22) Type III Pro-Gly-MetFBP21, PRP40 Type IV Phospho-Ser/phospho-Thr Pin1, Nedd4 *in directproximity to a proline-rich sequence (>50% proline content)

The transmutation of a WW domain from type I into one of type II, alongwith the consequent change in the specificity with reference to theproline-rich peptides, can be achieved, for instance, through the aminoacid exchanges L14W and H16G in the WW-type I-domain sequence. Thestructure of an agent from class I (Yes associated protein, YAP) showsthat this WW domain consists of three β-strands which form a β-sheet(see M. Macias, M. Hyvonen, E. Baraldi, J. Schultz, M. Sudol, M. Saraste& Mr. Oschkinat, The Structure of the WW Domain in Complex with aProline-Rich Peptide, Nature 382, pp. 646-649, 1996). The ligand bindingpocket is formed from the second β-strand of the beta sheet withcooperation from the second conserved tryptophan.

The most important biological role of WW domains evidently exists inintracellular signal transduction. WW domains furthermore have beenimplicated directly or indirectly with a number of diseases, forinstance inherited Liddle's syndrome, muscular dystrophy, and Alzheimersdisease; thus, they are the target of a series of therapeuticstrategies. Finally, WW domains play a biological role in the embryonicdevelopment of kidneys and in the intracellular life cycle ofretroviruses.

As part of this invention it was found that, astonishingly, WW domainscan form a stable structure (folding topology) under ordinary solventconditions, even if they are isolated from their original molecularcontext and are genetically fused in or, dependent on the situation, toother proteins, for instance a viral coat protein. This applies, forinstance, to a WW domain from the class of formin binding proteins withan unusually small size of only 31 amino acids, which forms a stablestructure (folding topology) under these conditions. Remarkably, theintroduction under favorable conditions of the WW domain, along withlinker segments consisting of the amino acids serine and glycine, intoexternal loops of proteins evidently neither disturbs their folding, norare the binding qualities of the WW domain thereby negativelyinfluenced. It could be shown that this also applies to variants of theWW domain, in which for instance amino acids were exchanged withcysteine at specific positions. It can also be valid for additionalstructures derived from WW domains, as for instance several consecutive,strung-together WW domains whose contributions to bonding eitherrespectively add up, or in favorable cases are synergistic, shortened orextended WW domains, or even WW domains with site-directed exchanges ofindividual amino acids which, dependent upon the desired application,can for example, bind more strongly or weakly at proline-rich sequencesthan the natural protein domains. Such altered domains can be obtained,for instance, through interaction screening by means of current phagedisplay technology.

Proteins which possess an inserted (that is to say, introduced insuitable loop regions within the polypeptide chain of the host proteins)or fused on (located respectively at the N-terminus and/or at theC-terminus of the host protein) WW domain demonstrate a high affinity toproline-rich sequences. These proline-rich sequences can thereby befused with other proteins, peptides, or other molecular substances. Twoarbitrary molecule species can therewith be brought into contact bymeans of the appended interaction partners (WW domain and proline-richsequence). This association rests first of all on a hydrophobicinteraction mediated by the WW domain and the proline-rich ligand. Thisinteraction can be adjusted, however, with regard to higher specificityand more flexible use, through enlistment of ionic interactions or theintroduction of covalent links between WW domain and proline-richligand, for example. Thus through placement of additional amino acidswhich are differently charged, or through point mutations in or near theadapter segments, the aggregation of the proline-rich ligands with theWW domain can be strengthened or more specifically configured. Acovalent disulfide bridging of both components allows, in turn, alasting and firm bond of the adapter segments and the molecularsubstances joined thereto.

A connection of more than two molecular substances with each other incompliance with the invention is also possible.

The kinetic parameters of interaction, such as the dissociation constant(k_(D)) could be determined as part of the investigations by interactionmeasurements based on surface plasmon resonance measurements. Withthese, it can be shown that the interactions between WW domains andproline-rich peptide sequence are fundamentally suitable for theapplications described in the invention at hand.

The nature of the interaction of adapter segments leads exclusively tothe development of a heteromeric hybrid species, consisting of a partwith WW domain and a part with a proline-rich sequence. The formation ofhomofunctional molecules (homodimers) can be excluded. Compared to othersystems with comparable characteristics, the utilized WW domain has theadvantage of being extraordinarily small and compact. Thereby it isclearly superior for many applications, for instance antigen antibodyinteractions, to other ligand binding domains (for example lipocalinsand anticalins).

Furthermore, it could be shown that the introduction of cysteineresidues at specific locations within the WW domain and in theproline-rich substrate can be used, beyond the interaction between WWdomain and proline-rich sequence, to cause covalent coupling of theassociation partner and as a result to bring together the protein partsfused onto the adapter segments in a stable connection. Thus, adissociation of the interaction partners can not result even underunfavorable conditions, for instance especially high or very low saltconcentrations, or under physiologically extreme temperatures. For that,an exchange with cysteine is undertaken for example at position Asp8(numbering follows the WW domain from the formin-binding protein FBP11)or alternatively at position Lys19. These positions are merely selectedexemplary; the introduction of specific cysteines can also be useful andsuccessful at other sites of the WW domain or the surroundings hereof,or in the proline-rich sequence or the surroundings hereof.

The particular advantage is that again only heterobifunctional specieswill be formed (heterodimers), since due to the strong interaction ofproline-rich peptide and WW domain, only associates between both ofthese two adapter segments can at first be formed. The subsequentdisulfide bridging under oxidizing conditions then leads to the directedformation of covalently bridged, heteromeric species. Due to the highlocal concentration (approximation) of cysteines in the associated form,the disulfide bridging can also be successful under slightly reducingconditions and can therewith take place with particular specificity. Incontrast, in case of accidental disulfide bridging, that is to saywithout the necessary strong affinity of the adapter segments to oneanother (that is, in a non patent-compliant application), undesirablehomodimers of both interaction partners would also be formed asbyproducts under oxidizing conditions.

The procedure described in the invention at hand is suitable to attacharbitrary interaction partners together in solution (in vitro), wherebyboth a temporary as well as a lasting link of both partners is possible.Likewise, the procedure can be used to specifically separate proteins,peptides or other molecular substances, those of which are equipped withone of the two adapter types (WW domain or proline-rich sequence), froma mixture of substances. This takes place through reversible binding toa matrix that has bound the respective interaction partners covalently.The strong bond is effective insomuch that the molecules also adhere tothe matrix under stringent solvent conditions. The process therebyallows, for instance, the fast and efficient purification of recombinantproteins from the crude cellular extract of bacteria or eukaryoticcells, on the condition that the recombinant (to be purified) moleculecarries one of the two adapter segments (WW domain or proline-richsequence) in fusion or as an insertion, while the correspondingcounterpart to the adapter segment is immobilized at the fixed phase.

Likewise, this immobilization procedure is suitable to implementspecific modifications or a refolding of the immobilized protein on thematrix, avoiding aggregation processes. Finally, with the invention athand, applications are also possible in which a simple and stableimmobilization of a molecular substance plays a key role, for instancein biosensors or in bioreactors (see R. S. Phadke, Biosensors and enzymeimmobilized electrodes, Biosystems 27, pp. 203-206, 1992; M.Abdul-Mazid, Biocatalysis and immobilized enzyme/cell bioreactors.Promising techniques in bioreactor technology, Biotechnology (N.Y.) 11,pp. 690-695, 1993).

Apart from proteins and peptides, other substances can be used for theprocedure described in the invention at hand. Thus peptide derivatives,peptide antibiotics, proteins with modified side chains such asfluorescence labels, alkylation, acetylation, disulfide mixtures withthiol-containing substances, and analogous changes can be deployed in asimilar manner. Peptide or protein conjugates with carbohydrate, nucleicacid or lipid content can also be utilized in the procedure. Nucleicacids such as DNA, RNA, ribozyme, synthetic nucleic acids such as, forexample, peptide nucleic acids, or hybrids thereof can likewise becoupled with an adapter segment, for example with chemical means. Theyare then likewise suitable to partake in an interaction with ananalogous interaction partner. The only requirement is the stableattachment of one of the utilized adapter segments.

Within the realm of invention-compliant application, antibodies,antibody-analogous substances, enzymes, structural proteins, andcapsomers of viruses or phages come in particular into consideration asproteins.

The insertion or attachment of proline-rich sequence or WW domain, or astructure derived therefrom, into or in a molecular substance can, inprinciple, take place at every site of the molecular substance, if thestructure of the WW domain is not substantially influenced thereby. Ifapplicable, it can be advantageous to implement the attachment orinsertion under utilization of suitable linker segments, as described inexample 1 for the protein PyVP1-WW150. In the case of insertion inproteins, it is expedient to seek out such areas of the proteinstructure in which no periodic secondary structural elements likeα-helix or β-sheet exist. The insertion of WW domains or proline-richsequences in protein structures takes place most favorably where turnareas or random coil areas of conventional definition exist.

The binding of both adapter segments to each other can be consideredunder the aspect of different physical interactions. Thus, a hydrophobiceffect can dominate the interaction during stabilization of theinteraction, as will be demonstrated in the following example 7. Otherforms of interaction can however also contribute to binding, such asionic interactions, ion-dipole interactions, dipole-dipole interactions,hydrogen bridge bonds, van der Waals forces, or dispersion forces.Ultimately, besides the aforementioned examples for non-covalentconnections, a covalent connection of both molecular substances can alsobe brought about. Thereby a chemically stable atomic bond between twoatoms of the interaction partners is created, preferably in the form ofdisulfide bridging of two participating cysteine side chains.

For immobilization of one of the adapter segments (WW domain orproline-rich sequence) the matrix can be charged, for instance, throughthe N-terminus of the proline-rich sequence or the WW domain (couplingthrough N-hydroxysuccinimide ester of the matrix) or through a thiolgroup of one of the cysteines contained in the proline-rich sequence orthe WW domain (coupling of the matrix through iodacetamide group). Asmatrices, for example, agarose and agarose derivatives, agarose beads,sepharose, dextrans, carbohydrates, or similar polymer material comeinto consideration based on current technology.

Applications of this invention are demonstrated in the followingexamples, through which the extent of protection of the invention shouldnot, however, be limited.

EXAMPLE 1 Insertion of a WW Domain in the Outer Segment of an In-VitroAssembled, Virus-Like Protein Coat (PyVP1-WW150)

In the first example, a WW domain of the amino acid sequenceGly-Ser-Gly-Trp-Thr-Glu-His-Lys-Ser-Pro-Asp-Gly-Arg-Thr-Tyr-Tyr-Tyr-Asn-Thr-Glu-Thr-Lys-Gln-Ser-Thr-Trp-Glu-Lys-Pro-Asp-Asp(SEQ ID NO:23) is inserted in a specific loop of a viral coat protein.At the same time, a linker is additionally inserted before and after theWW domain, consisting of alternating Gly-Ser amino acids. In the givenexample, the employed viral core protein is the pentameric polyoma virusVP1 core protein in solution, which based on current technology, iscapable of assembly in vitro into a virus-like coat. Based on thecrystal structure of the protein it can be recognized that a loop regionin the structure near to amino acid position 150 is possibly suitablefor the insertion of the WW domain, since this loop region is found onthe outside of the coat when assembly of the pentamer protein to avirus-like coat takes place.

The expression and purification of PyVP1-WW150 takes place as fusionprotein with a C-terminally fused intein domain and a chitin bindingdomain (CBD) connecting to that. For this, first of all, a plasmid isconstructed based on the vector pCYB2 of the IMPACT-System (New EnglandBiolabs). Through the multiple cloning site of pCYB2, with help of therestriction sites NdeI-XmaI (New England Biolabs), a DNA-fragment,coding for a variant of the VP1 gene of mouse polyoma virus, isamplified by PCR and cloned by standard methods.

As basis for this, a polyoma virus variant is used which displays nocysteines in the sequence whatsoever; the six cysteines of the wild typeprotein were previously replaced with serine with the help ofconventional mutation techniques. This variant of PyVP1 has theadvantage that the redox conditions of the solution have no influence onthe condition of the protein; it is thereby easily manageable in manyapplications. Additionally, by the later introduction of a cysteine inthe inserted WW domain, a specific disulfide bridge of WW domain andproline-rich sequence can be achieved. As a further variation, amodification to site 249 is used; the threonine found there in thewild-type protein is replaced with cysteine. At this site in theprotein, a marking with help of fluorescence dyes is, based on currenttechnology, advantageously possible. The protected localization in thepentamer allows the marking at this site without unwanted side-effects.The variant of polyoma virus VP1 used is correctly namedPyVP1-CallS-T249C, subsequently abbreviated with the term PyVP1.

For the PCR, the following oligonucleotides are used as primer: vp1NImp(5′-TAT ACA TAT GGC CCC CAA AAG AAA AAG C-3′; SEQ ID NO:24), and vp1CImp(5′-ATA TCC CGG GAG GAA ATA CAG TCT TTG TTT TTC C-3′; SEQ ID NO:25).With this PCR, the C-terminal amino acids of the wild-type VP1 proteinof Gly383-Asn384 are transformed simultaneously into Pro383-Gly384,since a C-terminally localized asparagine is very unfavorable regardingthe cleavage characteristics for the intein cleavage system. The pointmutations named do not further influence the essential characteristicsof the PyVP1 protein. The tac promotor of the pCYB2 vector provides onlyslight expression quantities of the fusion protein, thus the fusionconstruction PyVP1-intein-CBD is isolated from the pCYB2 vector throughan additional PCR, and cloned in the NdeI-EcoRI sites of ahighly-expressing pET-vector with T7lac promotor (plasmid pET21a,Novagen. Oligonucleotide:

vp1-NImp (5′-TAT ACA TAT GGC CCC CAA AAG AAA AAG C-3′), and 5′-ATA TGAATT CCA GTC ATT GAA GCT GCC ACA AGG-3′.

The cloning of the WW domain as insertion in the external Loop of PyVP1between the amino acid positions 148 and 149 takes place in severalsteps. With the oligonucleotides FBP11-WWaN (5′-ATA CTC TTC AGG CAG CGGCTG GAC AGA ACA TAA ATC ACC TGA TGG-3′; SEQ ID NO:27) and FBP11-WWaC(5′-ATA CTC TTC TAC CAC TAC CAT CAT CCG GCT TTT CCC AGG TAG ACT G-3′;SEQ ID NO:28), a PCR is implemented on a DNA-fragment which contains theformin-binding protein 11 (FBP11) from the organism Mus musculus(mouse). Among other things, a WW domain is encoded in this genesequence. The oligonucleotides simultaneously insert a short linkersequence of 5 amino acids apiece, consisting of alternatingglycine-serine amino acids. A second PCR on the previously describedvector amplifies the N-terminal fragment of PyVP1 between amino acids 1and 148 with help of the oligonucleotide vp1NImp (see above) andvp1-150-WWaC (5′-ATA CTC TTC AGG TAG CGG CGT AAA CAC AAA AGG AAT TTC CACTCC AG-3′; SEQ ID NO:29). Finally, a third PCR also amplifies theC-terminal fragment of PyVP1 between amino acids 149 and the C-terminalend of the protein, with help of the oligonucleotide vp1-150-WWaN(5′-ATA CTC TTC AGC CGC TGC CTG TAT CTG TCG GTT TGT TGA ACC CAT G-3′;SEQ ID NO:30) and vp1CImp (see above).

All three PCR products are subsequently digested with type IISrestriction enzyme EamI 104 I (Stratagene). The N-terminal and theC-terminal fragment of PyVP1 (PCR products 2 and 3, see above) aredephosphorylated with help of alkaline phosphatase (CIP, New EnglandBiolabs) to create a gene sequence in the following ligation step fromthe three prepared PCR-fragments, with the following sequence:(PyVP1-N-terminus)-WW domain fragment-(PyVP1-C-terminus). A PCRafterwards with the oligonucleotides vp1NImp and vp1CImp (see above)amplifies the ligation product of the three fragments, subsequentlyabbreviated with the term PyVP1-WW150. The PCR product can then becloned by means of the standard method in the vector pCR-blunt(Invitrogen). After cutting out the cloned fragment PyVP1-WW150 withhelp of the restriction enzyme Nde 1-Sma, the final cloning in thepreviously described plasmid pET21a can subsequently take place.

The lastly generated vector allows the expression of the fusion protein(PyVP1-WW150)-intein-CBD with help of highly expressive T7lac promotorsin E. coli BL21 (DE3) cells (mfr.: Novagen). For this purpose,transformed cells in 5 l.-Erlenmeyer flasks that contain 2 l. LB mediumeach, are cultivated at 37° C., until the OD₆₀₀ of the culture reaches2.0 to 2.5. Protein expression is induced by 1 mM IPTG in the medium.The cultures are thereafter incubated at 15° C. for a further 20 hours;the low temperature minimizes the cleavage of the intein part in thefusion protein under in vivo conditions. The cells are harvested bycentrifugation, dissolved in 70 ml resuspension buffer (20 mM HEPES, 1mM EDTA, 100 mM NaCl, 5% (w/v) Glycerol, pH 8.0), and lysed through highpressure homogenization. After centrifugation of the crude extract for60 min. at 48,000 G, a clear cellular extract is obtained. This extractis applied with a flow rate of 0.5 ml/min at a temperature of 10° C. ona 10 ml chitin affinity column (New England Biolabs). The column issubsequently washed with 3 column volumes of the resuspension buffer, 15column volumes of a wash buffer with high ionic strength (20 mM HEPES, 1mM EDTA, 2 M NaCl, 5% (w/v) glycerol, pH 8.0) and again 3 column volumesof the resuspension buffer; thereby all undesirable E. coli hostproteins are removed from the chitin matrix.

The cleavage of the PyVP1-WW150 monomer from the fusion protein by meansof self-cleaving intein activity is induced in resuspension bufferthrough a pulse (3 column volumes) with respectively 50 mMdithiothreitol (DTT), 50 mM hydroxylamine, or 30 mM DTT along with 30 mMhydroxylamine. For this, the loaded chitin matrix is incubated with oneof the indicated solutions for 14 hours at 10° C. The PyVP1-WW150protein is thereby completely released and can be separated, by means ofcolumn chromatographic standard methods, from the chitin matrix and theremaining elements of the fusion protein adhering to the matrix. Forthat purpose, a linear salt gradient is appropriately used with aconcentration between 0.1 and 2.0 M NaCl. The regeneration of the chitinmatrix takes place through washing of the chitin material with 3 columnsvolumes of an SDS-containing buffer (1% SDS (w/v) in resuspensionbuffer) according to manufacturer's instructions.

The PyVP1-WW150 protein is, in the described process, expressed as asoluble pentamer and is native. FIG. 2 a shows an SDS gel with thepurified fractions of wild type PyVP1 (or the variant PyVP1-CallS-T249Cderived therefrom) and of the PyVP1-WW150 variant, which exhibits ahigher mass because of the additionally inserted amino acids. FIG. 2 bshows comparable CD spectra of the produced proteins in 10 mM HEPES, 150mM NaCl, pH 7.2, which exhibit a correct folding of the protein species.A deconvolution of both CD spectra according to current technology showsthat in the case of the PyVP1-WW150 domain, an increase in the β-sheetstructure can be noted compared to that of the PyVP1 protein. Thisindicates that the inserted WW domain has kept its native structure asβ-sheet.

The example shows that surprisingly, the WW domain can be inserted withcorrect folding under suitable conditions in loop regions of proteinstructures, without substantially disturbing their native structure. ThePyVP1-WW150 protein, pentameric in solution, contains the native WWdomain inserted in the polypeptide chain and presents these, afterassembly, on the outside of the virus-like coat (see Example 2).

EXAMPLE 2 Characterization of the Properties of PyVP1-WW150

By means of example 1, a protein (PyVP1-WW150) can be produced whichartificially has a WW domain inserted. The binding qualities ofPyVP1-WW150 in relation to proline-rich ligands can be characterizedwith various processes. An advantageous method for this is provided bysurface plasmon resonance; in the given example, the instrument BiacoreX (Biacore AB) is used. A synthetic peptide of the sequenceCys-Ser-Gly-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Leu-Pro (SEQ ID NO:15) iscoupled, according to manufacturer's instructions via a thiol or aminocoupling, to a type CM5 sensor chip. In the process, initially aquantity of 80 resonance units of the indicated peptide (RU) isimmobilized on the surface. The following measurements always take placeat 25° C. and a flow rate of 20 μl/min.

Binding studies of PyVP1-WW150 at the sensor chip are carried out withimmobilized proline-rich peptide under various solvent conditions. Thefirst measurement takes place under standard solvent conditions, with 10mM HEPES, 1 mM EDTA, 150 mM NaCl, pH 7.2. The protein concentration ofPyVP1-WW150 is varied from 5 to 50 nM. In FIG. 3 a, it is apparent thatPyVP1-WW150 binds with high affinity to the sensor chip. The boundquantity is, as expected, proportional to the protein concentrationutilized. The binding constant K_(D) of the PyVP1-WW150 protein isdetermined thereby to have a value of 5 nM (FIG. 3 a). As is furthermoreobvious from the figure, the binding is not lasting, but the proteindissociates again in a slow process after the loading of the sensor'ssurface. This shows that the interaction of the interaction partners isreversible.

To test the binding under conditions of physiological ionic strength,Dulbecco's PBS (Gibco) is used as solvent for the second measurement.The remaining experimental conditions are selected analogously to thefirst experiment previously described. FIG. 3 b demonstrates thatbinding of the PyVP1-WW150 with Dulbecco's PBS displays no significantdifferences compared to binding under standard conditions (FIG. 3 a).From the experiment, binding parameters can be derived for theassociation (K_(on)=2105 M⁻¹s⁻¹) and dissociation (K_(off)=1.810⁻³ s⁻¹).The example shows that the changed solvent conditions have nosubstantial influence on the binding of PyVP1-WW150 to the proline-richpeptide, and suggests that the interaction of both partners also takesplace stably under physiological conditions. Thus, a fundamentalapplicability of the system under clinical conditions within the realmsof diagnostics or therapeutics is also possible.

For determination of the specificity of the binding, Dulbecco's MEMmedium with 10% FCS (fetal calf serum, Gibco) is used as running bufferin a third measurement. FCS here represents a model system for a mixtureof different proteins and other substances that are relevant inbiological systems. FIG. 3 c shows that also under these conditions asignificant and specific binding of the PyVP1-WW150 protein is to benoted at the sensor's surface. As in both previously describedmeasurements, the response signal at the sensor's surface is here alsoproportional to the concentration of PyVP1-WW150 protein introduced.Therewith, it is shown that the interaction of PyVP1-WW150 with theimmobilized proline-rich peptide is independent of the presence of amixture of other substances, as for instance occurs in serum.

In summary, these three analyses, with help of Biacore technology and asensor surface with immobilized proline-rich peptide, show that bindingbetween molecular substances that contain a WW domain and proline-richligands takes place with high affinity and specificity. The interactionis thereby reversible and not essentially dependent on the chosensolvent conditions. The dissociation takes place relatively slowlycompared to the association; the dissociation constant lies at 20 nM.

EXAMPLE 3 Immobilization on a Matrix

A further method for the characterization of binding qualities is areversible immobilization of the WW domain on an inert matrix. For this,a synthetic proline-rich peptide (sequenceCys-Ser-Gly-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Leu-Pro; SEQ ID NO:15) isconnected through a thiol coupling to SulfoLink column material (Pierce)according to manufacturer's instructions. A chromatography column ispacked with the matrix modified in this way. This permits a loading ofthe samples on the matrix and elution with bound proteins underdifferent conditions. The PyVP1-WW150 protein purified as in example 1is applied to the column (solvent 10 mM HEPES, 1 mM EDTA, 150 mM NaCl,5% Glycerin, pH 7.2). As evident from FIG. 4, the protein binds to thematrix and appears only in slight quantities in the wash fractions. Asubsequent elution of the protein from the matrix is possible throughaddition of 1% SDS or 300 mM arginine to the running buffer.

This experiment shows that the PyVP1-WW150 protein is able to reversiblybind to a matrix that carries a proline-rich peptide. An temporaryimmobilization can thereby take place. A detachment of the protein fromthe matrix is possible through use of additives in the running buffer.

EXAMPLE 4 Binding of a Proline-Rich Peptide to a Capsid

In a further experiment, the binding of a fluorescence-marked peptidewith a proline-rich sequence to the surface (exterior) of virus-likecapsids is explored. The assembly of the protein takes place in analogyto conditions already described, based on current technology (seeSalunke, Caspar & Garcea, Polymorphism in the assembly of polyoma viruscapsid protein VP1, Biophys. J. 56, pp. 887-900, 1989). The virus-likecapsids are obtained after dialysis of the protein against 10 mM HEPES,50 mM NaCl, 0,5 mM CaCl₂, 5% Glycerin, pH 7.2. The proline-rich peptideCys-Ser-Gly-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Leu-Pro (SEQ ID NO:15) islabeled specifically at the N-terminal cysteine with afluorescein-maleimide derivative (Molecular Probes) according tomanufacturer's instructions. After the assembly into capsids of thevirus protein variants, a tenfold molar excess of fluorescence-markedpeptide is added. Through gel filtration (column TSKGel G5000PWXL,TosoHaas), virus-like capsid coats can be clearly detected and separatedfrom free, non-assembled capsid elements as well as from surplus peptideand fluorescence dye. The peptide bound to the WW domain located on thesurface of the capsid elutes in the capsid fractions and can be verifiedthrough the specific absorption of the fluorescence dye (FIG. 5).

This example shows that the PyVP1-WW150 variant can form capsidstructures (virus-like coats) under suitable conditions. These capsidsare able to bind proline-rich peptide. Thus, molecular substances can bebrought in a directed manner to the surface (exterior) of virus-likestructures via the specific and strong interaction of WW domain andproline-rich sequence.

EXAMPLE 5 Packaging of GFP in the Interior of a Virus-Like Protein Coat

In this example, it is shown that through favorable positioning ofadapter segments, a localization of molecular substances into theinterior of viral coats, or of virus-like coats (capsids) can takeplace. Due to the three-dimensional structure of polyoma virus VP1 it isknown, based on current technology, that the N-terminus of the proteinis localized in the interior of the coat after assembly into the capsid.The first 14 amino acids of the protein are thereby possiblyunnecessary, since they can not be detected in the x-ray structure ofthe capsids. Hence two different variants of the PyVP1 protein areproduced, which contain a WW domain at the amino terminus of the nativewild-type protein (variant PyVP1-3C-WW1) or carry the WW domain at anN-terminus shortened by 14 amino acids (variant PyVP1-3C-WW[N-14]), aswell as a variant of the PyVP1 protein which carries a proline-richsequence at the N-terminus (PyVP1-3C-[N-14]-PLP). The basis for thesevariants is a PyVP1 variant which contains the cysteines C19 and C114,and in which a specific new cysteine is additionally introduced(analogous to the variant PyVP1-CallS-T249C). This variant is hereafterabbreviated with PyVP1-3C.

First of all, an amplification of the WW domain is performed by means ofPCR; the FBP11 gene of the mouse serves thereby as a template, analogousto example 1. As oligonucleotide for the PCR, 5′-AAT ATA TCA TAT GTC CATCAT CCG GCT TTT CCC AGG TAG ACT-3′ (SEQ ID NO:31) (with NdeI interface),and 5′-TAT TAA TCA TAT GAG CGG CTG GAC AGA ACA TAA ATC ACC TGA TGG-3′(SEQ ID NO:32) are thereby used. The PCR product obtained issubsequently cloned, through the cutting sites Nde I-Nde I introduced bymeans of the oligonucleotide, into the expression vector pET21a fromexample 1, which contains the gene for a fusion proteinPyVP1-intein-CBD; at that 5′ end of the gene, a singular Nde Irestriction site is found. The expressed gene product of this vector isthe desired protein PyVP1-3C-WW1. Analogous thereto, cloning with afragment of PyVP1-3C shortened by 14 amino acids (PyVP1-3C-WW[N-14]) isperformed based on the standard method described in example 1. To thisend, a PCR is performed on the PyVP1 genetic fragment, with 5′-GCG CGCGCA TAT GAG CAC CAA GGC TAG CCC AAG ACC CG-3′ (SEQ ID NO:33) and theoligonucleotide vp1CImp (see Example 1). The PCR product that results isdigested with the restriction enzymes Nde I-Sma I, and the fragmentcloned into the vector pET21a from example 1 using the standard method.Expression and purification of both proteins takes place in accordancewith example 1. The purified proteins are compared to the variants PyVP1and PyVP1-WW150 in FIG. 6 a. It shows that the proteins are soluble andnatively producible. The change of the N-terminus through theintroduction of the WW domains has no important negative influence onthe assembly competence of the protein for the creation of virus-likeshell structures.

In an analogous manner, the manufacture and purification of a GFPvariant is undertaken. GFP is a protein that displays a greenfluorescence (absorption maximum at 490 nm) in the native condition. Itis excellently suited for the marking of complexes and assemblies. Forthe manufacture of a GFP variant with a proline-rich terminal sequence,a PCR-based amplification of the GFP gene first of all is carried out,with the plasmid pEGFP-N1 (Clontech) serving as template.Simultaneously, suitable restriction sites are introduced into the PCRproduct. The PCR takes place by means of the oligonucleotides 5′-TTA TTTACA TAT GGT GAG CAA GGG CGA GGA G-3′ (SEQ ID NO:34) (with Nde I-cuttingsite) and 5′-ATA TCT TAA GTA CAG CTC GTC CAT GCC G-3′ (SEQ ID NO:35)(with AflII cutting site). The PCR product so obtained is cloned, overthe restriction site, into the vector pTIP and expressed there. Thisvector pTIP is a derivative of the intein purification vector documentedin example 1, on basis of pET21a, with additionally introducedproline-rich sequences. The vector is constructed so that a proline-richsequence can be selectively fused to the 5′ or 3′ ends of a geneintegrated in a multiple cloning site. The proline-rich sequence therebycontains mainly Pro-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Leu-Pro (SEQ ID NO:36).The manufacture and purification of the GFP-PLP protein takes place bymeans of chitin affinity chromatography, in accordance with the methodin example 1. The successful manufacture and purification of GFP-PLP isdocumented in FIG. 6 b. The GFP-PLP protein, which carries theproline-rich sequence Pro-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Leu-Pro (SEQ IDNO:36) at the C-terminus, can be produced in solution in large quantity.The green luminous color of the protein solution simultaneously showsthat the protein can fold to its native structure.

The manufacture of the Py-VP1-3C-PLP variant takes place analogously.For manufacture of this PyVP1 variant, PyVP1-3C-[N-14] is cloned intothe vector pTIP, so that the the proline-rich sequence contained in thevector is fused N-terminally to the Py-VP1-3C-[N-14].

For inspection of the functional characteristics of both PyVP1 variantswith WW domain at the respective N-terminus of the proteins, bothvariants are incubated with proteins that contain proline-richsequences. The PyVP1-3C-WW1 protein is incubated with the prior-producedprotein GFP-PLP (molar relationship 1:6) for 10 min. (10 mM HEPES, 1 mMEDTA, 150 mM NaCl, 5% Glycerol, pH 7.2), and the capsid formation of thePyVP1 variants induced through dialysis against a buffer which contains0.5 mM CaCl₂ (see Example 4). The successful detection of capsids (FIG.7 a) demonstrates that the variant PyVP1-3C-WW1 is competent forassembly under suitable conditions. Using gel filtration assays (columnTSKGel G6000PWXL, TosoHaas), it can be shown that a slight portion ofthe native GFP-PLP protein (identifiable by the specific absorption of490 nm) is contained in the capsid fraction (at elution volumes between9 and 10 ml) (FIG. 7 a). That means that during the incubation ofGFP-PLP protein with the PyVP1-3C-WW[N-14] variant a binding of bothproteins to one another takes place, whereby the GFP was directed intothe interior the virus-like of particle during the subsequent capsidassembly.

EXAMPLE 6 Packing of a Peptide in the Interior of a Virus-Like ProteinCoat

In a second experiment analogous to experiment 5, PyVP1-3C-WW[N-14] isincubated in a molar relationship of 1:10 with a proline-rich peptidethat was previously fluorescence-labelled. The labeling of the peptide(Cys-Ser-Gly-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Leu-Pro; SEQ ID NO:15) heretakes place by means of fluorescein maleimide (Molecular Probes) througha maleimide coupling of the dye to the N-terminal cysteine, according tomanufacturer's instructions. Again, as in the example 5, it is shownthat after assembly of the PyVP1-3C-WW[N-14] variant, PyVP1-3C-WW[N-14]is competent for assembly under suitable conditions. In addition, theprotein is able to bind the proline-rich peptide and, during theassembly into a protein coat, to bring the proline-rich peptide into theinterior of the capsid. This is shown with the gel filtration in FIG. 7b through the specific absorption at 490 nm of the fluorescence dyebound to the peptide covalently, which is found mainly in the elutionregion of the capsid (9 to 10 ml).

Moreover, using the variants of PyVP1-3C-[N-14]-PLP (proline-richsequence at the N-terminus) and GFP-WW1 (WW domain at the N-terminus),an analogous assembly attempt can be carried out. This shows that areciprocal disposition of WW domain and proline-rich ligands at thesubstances to be joined, that is, the placement of proline-rich sequenceon the polyoma core protein (capsid) and the WW domain on the protein tobe wrapped, also leads to a successful targeting of GFP into theinterior of the virus-like coat.

Summing up, the experiments in examples 5 and 6 show that variants ofPyVP1 with WW domain fused to the N-terminal are able to bindproline-rich sequences and to conduct these, as well as any molecularsubstances located on them, under suitable conditions into the interiorof virus-like coats during assembly into capsids. The described processis thereby suitable to cause a directed wrapping of molecular substancesin viruses or in virus-like capsids. It could likewise be shown thatvariants of PyVP1 with a proline-rich sequence fused to the N-terminalare able to bind WW domains and molecular substances located on them.

EXAMPLE 7 Disulfide Bridging for Covalent Linkage on the Basis ofModified WW Domains and Proline-Rich Peptides

The investigations described in the previous examples 1 to 6 show thatthrough the interaction of WW domain with a proline-rich peptidesequence, a temporally limited aggregation of both adapter segments canoccur. A permanent bridging of interaction partners can be achieved byequipping both adapter segments with specifically-introduced cysteineamino acids, which allow, with suitable positioning, disulfide bridgingafter the association of the binding partners.

Through point mutations, performed in accordance with conventionalprocesses based on current technology, individual amino acids that arenot essential for the association of both adapter segments can bechanged to cysteines, both within the WW domain as well as in theproline-rich sequences of the ligands. Under suitable redox (oxidizing)conditions, a specific disulfide bridging can be formed between thebound proline-rich ligands and the WW domain, each containing one ormore cysteines. This bridging is thereby decisively favored through theinteraction of WW domain and the ligands. The temporally limitedinteraction between unconnected WW domain and proline-rich ligands lastslong enough to form a covalent linkage through disulfide bridging. Theinteraction of both adapter segments becomes, in this way, temporallyunlimited, since disulfide bridges under physiological conditions, asthey exist for instance in extracellular space, are stable. If desired,the disulfide bridge between WW domain and proline-rich ligands can beeliminated again in vitro, under reducing conditions (for instance 50 mMDTT, DTE or -Mercaptoethanol); by removal of the reducing agent, areconnection is also possible.

Based upon the variant PyVP1-WW150 of the core protein VP1 of murinepolyoma virus, an aspartate amino acid (position 8 into the WW domain)is transformed through mutagenesis into a cysteine. The resultingcysteine-containing variant is subsequently named PyVP1-WW150-D8C.Through binding studies based on surface plasmon resonance, it can beshown that this variant of the WW domain binds the proline-rich ligandsCys-Ser-Gly-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Leu-Pro (SEQ ID NO:15), evenwithout creation of disulfide bridging. The extent of the interactionis, however, somewhat slighter than with PyVP1-WW150. This can evidentlybe ascribed to the newly introduced cysteine. It can be shown that thisaccumulation can be strengthened through addition of 500 mM ammoniumsulfate. Thereby, the hydrophobic interactions between proline-richligands and WW domain are presumably intensified. The intensity of theinteraction can thus be modulated through the ammonium sulfateconcentration in the solvent.

The formation of the disulfide bridge between proline-rich ligands andWW domain subsequently takes place under slightly oxidizing conditions.For this purpose a buffer is used which both contains ammonium sulfateand maintains defined redox conditions. The latter conditions areachieved through usage of 1 mM GSSG and 5 mM GSH in the redox buffer (50mM Tris, pH 8.5, 1 mM EDTA, 500 mM ammonium sulfate); oxidized (GSSG) orreduced glutathione (GSH) functions thereby as a redox shuffling systemfor the formation of disulfide bridges (see R. Rudolph, In vitro foldingof inclusion body proteins, FASEB J. 10, 49-56, 1996). The disulfidebridging is carried out at 15° C. for 24 h and completed throughdialysis against 50 mM Tris, 1 mM EDTA, pH 7. Under the conditionsmentioned last, no further disulfide exchange occurs; the formeddisulfide bridges are stable.

Summing up, it can be said that the introduction of cysteine amino acidresidues into the WW domain make possible the covalent bridging ofpolyproline-rich ligands which carry at least one cysteine, with the WWdomain and thereby lead to a stable covalent linkage of WW domain andligand (see FIG. 9).

EXAMPLE 8 Purifying of Proteins by Means of Adapter Segments(Polyproline/WW-Affinity Chromatography)

A further area of application of the invention at hand is the separationof molecular substances from mixed substances, as is typically done inthe purification of proteins from crude extracts (cellular extracts). Inthe process, the affinity of the WW domain to proline-rich ligands isexploited to isolate proteins that contain a WW domain from a complexmixture (crude extract) of proteins (principle of affinitychromatography). For this purpose, a column is used as in example 3; theSulfoLink material (Pierce, the reactivity of the matrix with SH groupsis based on the iodacetamide group at the end of a linker, consisting of10 CH₂-groups) is thereby loaded through a thiol coupling with thepeptide Cys-Ser-Gly-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Leu-Pro (SEQ IDNO:15), according to manufacturer's instructions.

Analogously, the coupling of peptides to other matrices is possible, forinstance AffiGel 10 (Biorad, the reactivity of the matrix with NH₂groups is based on the N-hydroxysuccinimide group at the end of alinker, consisting of 10 CH₂-groups) through the N-terminus of thepeptide. Likewise, the peptide coupling can take place at the N-terminusof the peptide to a matrix based on CH-sepharose 4B, (Sigma, thereactive group of the matrix is likewise an N-hydroxysuccinimide ester).A covalent binding of the proline-rich ligands to a carrier materialalso results here, which subsequently allows a purification of WW domainproteins.

The PyVP1 variant PyVP1-3C-WW1 from example 5 (WW domain at theN-terminus of the PyVP1 protein) is, analogous to the specificationsfrom example 1, produced as a fusion protein with an intein and achitin-binding domain ([PyVP1-3C-WW1]-intein-CBD). It is not, however,purified with the described standard method, by means of chitin-affinitychromatography. Instead, the cellular extract, after cellular breakdownand subsequent centrifugation, is applied to the previously-describedcolumn. 10 mM HEPES, 150 mM NaCl, 1 mM EDTA, 5% Glycerin, pH 8.0 servethereby as running buffer. After application of the extract, it iswashed with 10 column volumes of a buffer which contains 10 mM HEPES (pH8.0), 1 mM EDTA, 5% Glycerin, and in addition 2 M NaCl. With this washprocedure, all non-specific adsorbed proteins and cellular componentsare removed from the SulfoLink matrix. Afterwards, elution of the boundfusion protein [PyVP1-3C-WW1]-intein-CBD takes place with a buffer whichcontains 2% SDS. As can be recognized in FIG. 8, a binding of the fusionprotein [PyVP1-3C-WW1]-intein-CBD takes place through the interaction ofthe WW domain with the immobilized proline-rich peptide in the SulfoLinkmatrix. The fusion protein is thereby bound to the matrix and thus mostof the other proteins of the cellular extract are removed inflow-through or during the wash process. Subsequently, the elution withSDS almost exclusively delivers the complete WW domain-containing fusionprotein, as well as proteolytic degradation products thereof (which inthe case of PyVP1, appear in all comparable manufacture processes basedon current technology) and molecular chaperones, which, as is commonlyknown, are able to bind directly to PyVP1 and cannot usually beseparated. Instead of elution of the bound WW domain protein by means ofSDS, an elution of native protein with 300 mM arginine in the runningbuffer is also possible. Through subsequent dialysis of the eluate forremoval of the arginine, one receives the purified, native protein.

In summary, this example shows that with the described system it ispossible to separate and purify specific molecules from a mixture ofsubstances (crude extract).

EXAMPLE 9 Specific Dimerization of Molecules Through Adapter Segments

Using the interaction of adapter segments (WW domain and proline-richpeptide), the manufacture of bifunctional or bivalent hybrid moleculesin vitro can also take place. To that end, two molecular substances areproduced which, according to application, can have identical ordifferent characteristics, and each of which always carries one of theadapter segments covalently linked. In the chosen example, themanufacture of easily-detectable dimers of the GFP protein is performed.

A variant of GFP is produced for this purpose, analogous to theproduction of PyVP1 and with help of the intein-based expression systemfrom example 1, with a WW domain at the N-terminus of the GFP (GFP-WW1).First of all, a PCR on the vector pEGFP-N1 (Clontech, see Example 5) isimplemented with the oligonucleotides 5′-TAT AGC TAG CGT GAG CAA GGG CGAGGA GCT GTT C-3′ (SEQ ID NO:37) and 5′-GGG AAT TAA GTA CAG CTC GTC CATGCC G-3′ (SEQ ID NO:38). The PCR product is ligated through the cuttingsites Nhe I-Sma I into the vector pET21a from example 5, which, at the3′ end of the insertion site, contains the fusion protein of intein andchitin binding domain (CBD) described in example 1. On the 5′-side ofthe insertion site, the WW domain that is described in example 5 isfound. Thus, a fusion product is made with the sequence WWdomain-GFP-intein-CBD. The plasmid that encodes the fusion protein canbe transformed into the E. coli strain BL21(DE3). Analogous to examples1 and 3, the manufacture and purification of fusion proteins can thentake place. With the described process, the protein GFP-WW1 can beproduced in purified form.

A second variant of GFP is produced analogously to this, with aproline-rich segment (Pro-Pro-Pro-Pro-Pro-Pro-Pro-Pro-Leu-Pro; SEQ IDNO:36) at the C-terminus. The manufacture and purification of theGFP-PLP protein thereby takes place identically to the description forthe protein in example 5.

Both GFP variants are subsequently incubated together. Both proteins arethereby brought into connection with each other through the adaptersegments and as a result a GFP dimer is formed that can be distinguishedfrom the GFP monomer through gel filtration in a TSK-PW2000XL gelfiltration column (TosoHaas).

The example demonstrates that with help of the procedure described inthe invention at hand, a connection of arbitrary molecular substancescan take place, which carry appropriate adapter segments on the basis ofWW domains or proline-rich peptide segments. Homofunctional orheterofunctional assemblies can thereby be formed.

EXAMPLE 10 Packaging of WW Domain Containing Peptides into the Interiorof a Virus-Like Shell

In this example it is shown that a direct packaging of proteins(GFP-WW1) which contains a WW domain can be achieved by positioning aproline-rich sequence at a protein shell. This example therefore showsin a mirror fashion to the elaborately documented example 5 in thispatent application that the WW domains rsp. proline-rich sequences usedas anchoring molecules are interchangeable. The result of thisexperiment was already represented in a short form in the patentapplication (FIG. 7 b, and figure legend to FIG. 7 in the patentapplication).

For the production of the expression plasmid for the GFP-WW1 fusionprotein the vector pyVP1-3C-WW1 described in example 5 in the patentapplication is used, which contains a NheI restriction site in the VP1gene close to the 5′ end. The GFP gene was amplified by PCR using theoligonucleotides 5′-TAT AGC TAG CGT GAG CAA GGG CGA GGA GCT GTTC-3′(NheI) and 5′-GGG AAT TAA GTA CAG CTC GTC CAT GCC G-3′(SmaI),thereby introducing a NheI cutting site at the 5′ end and a SmaI cuttingsite at the 3′ end. The VP1 gene in the vector pyVP1-3C-WW1 is replacedby the GFP gene, thereby creating an open reading frame for the fusionprotein GFP-WW1.

In an analogous way, the production of the VP1 coat protein with anN-terminally fused proline-rich sequence is performed. First, the VP1gene from the vector pyVP1-3C-WW1 is amplified using PCR, whereby anAflIII restriction site is introduced by the oligonucleotide 5′-TAT ACTTAA GTA CAA AGG CTT GTC CAA GAC CCG C-3′ and an EcoRI restriction siteis introduced using the oligonucleotide 5′-ATA TGA ATT CCA GTC ATT GAAGCT GCC ACA AGG-3′. Subsequently, the VP 1 gene is inserted into thevector pTIP described in example 5 of the patent application whichresults in an N-terminal fusion of the proline-rich sequence to VP1.

The production and purification of the fusion proteins described beforeoccurs by chitin affinity chromatography in analogy to the proceduredescribed in example 1 of the patent application.

In order to check the functional properties of the two proteins, theyare incubated together and the association of the proteins via theiradapters is analyzed using gel filtration chromatography. For this, theGFP-WW protein is added in a fivefold molar excess to the VP1-3C-PLPprotein and subsequently incubated for 10 min in a buffer in which theVP1 is contained in its pentameric form, and therefore the accessibilityof the adapters is ensured (pentamer buffer 10 mM HEPES, 1 mM EDTA, 150mM NaCl, 5% Glycerol). Subsequently, the capsid formation is induced byaddition of 0.5 mM CaCl2. Gel filtration analysis (column TSK GelG6000PWXL) demonstrate that the VP1-3C-PLP variant is assembly competentunder suitable conditions. In addition, it is demonstrated that GFP-WW1is contained in the capsid fraction (FIG. 7 b of the patentapplication). Therefore, during the incubation over 10 minutes describedbefore, an association between the GFP-WW1 and the VP1-3C-PLP proteinhas occurred by their adapters, so that the GFP-WW1 was directeddirectly into the interior of the virus-like particles during thefollowing capsid formation.

The example shows therefore that WW domain and proline-rich sequence canbe introduced in different molecular environments, respectively, and themolecular substances modified in this way can so be linked together.Additionally, in the present special case of this example one of themolecular substances is an assembly-competent capsid, so that moreover adirection of the molecular substance into a protein shell was possible.

1. Method for linking of two or more molecular substances with eachother through adapter segments, said method comprising: (a) modifyingone of the molecular substances in such a way that it exhibits as anadapter segment, in at least one region, a WW domain, (b) modifyinganother molecular substance in such a way that it exhibits as an adaptersegment, in at least one region, a proline-rich sequence, which binds tothe WW domain, and (c) forming a covalent bond between the molecularsubstances by interaction between said WW domain and said proline-richsequence, wherein said molecular substances are members selected fromthe group consisting of antibodies, substances analogous to antibodies,enzymes, structural proteins, peptide antibiotics, isolatedstructure-forming, catalytic, or regulatory protein domains, fragmentsor proteins, peptides, peptide analogs, antigen-bearing substances,glucoproteins, lipoproteins, and proteoglycans.
 2. Method for linking oftwo or more molecular substances with each other through adaptersegments, said method comprising: (a) modifying one of the molecularsubstances in such a way that it exhibits as an adapter segment, in atleast one region, a WW domain, (b) modifying another molecular substancein such a way that it exhibits as an adapter segment, in at least oneregion, a proline-rich sequence, which binds to the WW domain, and (c)introducing one or several cysteines in the region of the WW domain andone or more cysteines in the region of the proline-rich sequence, andforming a covalent link between said molecular substances at saidcysteines, wherein said molecular substances are members selected fromthe group consisting of antibodies, substances analogous to antibodies,enzymes, structural proteins, peptide antibiotics, isolatedstructure-forming, catalytic, or regulatory protein domains, fragmentsor proteins, peptides, peptide analogs, antigen-bearing substances,glucoproteins, lipoproteins, and proteoglycans.
 3. Method according toclaim 1 or 2, wherein one of the molecular substances is a solid phasematrix molecule.
 4. Method according to claim 1 or 2, wherein the WWdomain is found in a loop region of a protein structure so modified or aC- or N-terminus of a protein or peptide structure so modified. 5.Method according to claim 1 or 2, wherein the proline-rich sequence isfound in a loop region of a protein structure so modified or a C- orN-terminus of a protein or peptide structure so modified.